The invention relates to a condensing enzyme involved in long chain fatty acid production in plants, including related nucleic acid sequences.
Living organisms synthesize a vast array of different fatty acids, which are incorporated into complex lipids. These complex lipids represent both major structural membrane components, and are a major storage product in both plants and animals. In plants, very long chain fatty acids (VLCFAs, chain length C20 or longer) are synthesized predominantly in the epidermal cells where they are either directly incorporated into waxes, or serve as precursors for other aliphatic hydrocarbons found in waxes, including alkanes, primary and secondary alcohols, ketones, aldehydes and acyl-esters (for review see Post-Beittenmiller, 1996). VLCFAs also accumulate in the seed oil of some plant species, where they are incorporated into triacylglycerols (TAGs), as in the Brassicaceae, or into wax esters, as in Jojoba. These seed VLCFAs include the agronomically important erucic acid (C22:1), that may be used in the production of lubricants, nylon, cosmetics, pharmaceuticals and plasticizers.
VLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system which involves four enzymatic reactions: (1) condensation of malonyl-CoA with a long chain acyl-CoA, (2) reduction to beta-hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the acyl-CoA elongated by two carbons. The condensing enzyme catalyzing reaction (1) is a key activity of the FAE system. It is the rate-limiting enzyme of the VLCFA biosynthetic pathway, which controls the amount of VLCFAs produced (Miller and Kunst, 1997). In addition, the condensing enzyme determines the ultimate VLCFA acyl chain length, and thus their uses in seed oil or wax biosynthesis.
Different condensing enzymes acting on, and producing, acyl chains of different length have recently been characterized. Several groups independently identified the first plant fatty acid elongation gene in Arabidopsis, FAE1 (James and Dooner, 1990; Kunst et al., 1992; and Lemleux et al., 1990). FAE1 was subsequently cloned and is described in WO9613582 as catalyzing the conversion of C18 fatty acids to C20-C22 fatty acids. The patent WO9613582 suggests that FAE1 will have activity in a very broad host range. In support of this assertion of broad host range activity, it has been shown that FAE1 has the same activity in yeast as in Arabiqopsis (Miller and Kunst, 1997). A Jojoba protein involved in the synthesis of VLCFAs has also been isolated having relatively high homology to FAE1 (52% amino acid identity), and has been shown to have beta-ketoacyl-coenzyme A synthase (KCS) activity (WO9515387). Broad host range activity for genes encoding KCS has been further evidenced by the Jojoba KCS cDNA. The Jojoba KCS cDNA was able to complement a mutation in a Canola variety of rapeseed (Brassica napus), to restore in the variety high levels of VLCFAs (Lassner et al., 1996). An Arabidopsis gene (CUT1), required for cuticular wax biosynthesis and pollen fertility, has also been described as encoding a VLCFA condensing enzyme that catalyzes the addition of 2C units to pre-existing C24 or longer fatty acids (Millar et al., 1999).
The broad specificities exhibited by FAE activities provide a means of modifying the synthesis of VLCFAs in a given cell. As evidenced by the accumulation of VLCFAs in tobacco seed expressing FAE1 (Millar and Kunst, 1997), heterologous condensing enzymes may be used to produce VLCFAs in plant species that do not otherwise synthesize VLCFAs. For example, targeted expression of heterologous VLCFA condensing enzymes in seeds may allow the production of crop plants capable of synthesizing VLCFAs of desired lengths in seed oil for industrial applications. New condensing enzymes may also be useful for the manipulation of cuticular waxes which have important functions in many physiological processes in plants, including water balance, protection from UV light, plant-insect interactions, and defense against bacterial and fungal pathogens (Post-Beittenmiller, 1996). As a result, there is a need for new condensing enzymes that may be used alone or in combination with other condensing enzymes to confer on plants and plant tissues the ability to synthesize a range of VLCFAs, including VLCFAs up to C30 in length.
There is also a need for tissue-specific promoters capable of mediating the expression of heterologous condensing enzymes in epidermal cells, which may facilitate the alteration of the wax composition and/or accumulation in plants. This may, in turn, result in the production of crops with increased tolerance to environmental stresses, and/or resistance to pathogens and insects. For example, drought resistance in rice is associated with high wax lines rich in C29, C33 and C35 alkanes (O""Toole and Cruz, 1983; Haque et al., 1992). Increased wax deposition may also be accomplished by overexpression of condensing enzymes with desired acyl chain length specificities using an epidermis-specific promoter, such as the CUT1 promoter (Millar et al., 1999).
The cumulative data, as discussed, from a variety of sources in this field has led to the suggestion that the amounts and acyl chain lengths of VLCFAs, in a wide variety of eukaryotic cells, are regulated by the nature of condensing enzyme expression in the cell (Miller and Kunst, 1997). Condensing enzymes would therefore be useful in a range of biotechnical applications.
In various aspects, the present invention provides nucleic acid sequence encoding all or part of a new plant long chain fatty acid condensing enzyme (fatty acid elongase), designated herein as KCS2 (for beta-ketoacyl-coenzyme A synthase 2). In some embodiments, KCS2 may mediate the biosynthesis of C18, C20, C22 and C24 fatty acids. The activity of the enzyme is typically characterized by two carbon (malonyl-CoA) additions to C16, C18, C20 and C22 moieties (C16-C22 acyl CoA molecules), i.e. condensation of malonyl-CoA with a C16, C18, C20 or C22 acyl-CoA. The fatty acids produced by the enzyme may for example be saturated 18:0, 20:0, 22:0 and 24:0 fatty acids.
The invention includes recombinant nucleic acid molecules comprising a heterologous nucleic acid coding sequence encoding the plant long chain fatty acid condensing enzyme. In alternative embodiments, the nucleic acid coding sequence may be derived from the Arabidopsis KCS2 coding sequence disclosed herein. Alternatively, embodiments include nucleic acids that encode the plant very long chain fatty acid condensing enzyme of the invention, wherein the enzyme has an amino acid sequence that is at least 70% identical to an Arabidopsis KCS2 amino acid sequence disclosed herein, when optimally aligned. The nucleic acid coding sequences of the invention also include sequences that hybridize under stringent conditions to a complement of the Arabidopsis KCS2 coding sequence disclosed herein. The nucleic acid coding sequences of the invention may also be at least 70% identical to the Arabidopsis KCS2 coding sequence, when optimally aligned. Embodiments of the invention include isolated nucleic acid molecules comprising the coding sequences of the invention.
In another aspect, the invention provides recombinant nucleic acid molecules comprising a promoter sequence operably linked to a nucleic acid sequence, wherein the promoter sequence is capable of mediating gene expression in anthers and in very young leaves in Arabidopsis. The promoter sequences of the invention may be derived from an Arabidopsis KCS2 promoter sequence, as disclosed herein. Promoter sequences of the invention may also hybridize under stringent conditions to the Arabidopsis KCS2 promoter sequence disclosed herein. Promoter sequences of the invention may also be at least 70% identical to the Arabidopsis KCS2 promoter sequence when optimally aligned.
The invention provides nucleic acid probes comprising probe sequences that hybridize under stringent conditions to a portion of an Arabidopsis KCS2 genomic sequence; or are at least 70% identical to the portion of an Arabidopsis KCS2 genomic sequence when optimally aligned. The invention also provides methods of isolating a nucleic acid molecule encoding a plant long chain fatty acid condensing enzyme, for example by hybridizing a nucleic acid preparation with the nucleic acid probe of the invention.
The invention provides transgenic cells (such as plant cells), plants and parts thereof, in which the plants or cells comprise the recombinant nucleic acid molecules of the invention. Such plant parts may for example include seeds or oils. Transgenic plants of the invention may have a modified phenotype compared to a non-transgenic plant of the same species, such as a modified lipid content. Methods of producing such transgenic plants are provided, for example by introducing into a plant the isolated nucleic acids of the invention. Progeny plants may be provided, produced by sexual or asexual propagation of the transgenic plants of the invention to produce transgenic descendants of transformed plants.
Purified proteins are provided, encoded by the recombinant nucleic acid molecules of the invention, including plant fatty acid condensing (elongase) enzymes and fragments thereof. Also provided are recombinant vectors comprising the recombinant nucleic acid molecules of the invention.
Antisense nucleic acid molecules are provided, wherein a portion of the nucleic acid coding sequences of the invention are provided in reverse orientation relative to an adjacent promoter sequence. Recombinant antisense nucleic acids of the invention may therefore encode an antisense RNA that hybridizes under stringent conditions to a complement of a portion of the Arabidopsis KCS2 coding sequence; or that are at least 70% identical to a portion of the Arabidopsis KCS2 coding sequence when optimally aligned. Transgenic plants or plant cells of the invention may include the recombinant antisense nucleic acids of the invention.
In one embodiment, the nucleic acid coding sequences of the invention may be substantially identical to all or part of an Arabidopsis KCS2 coding sequence, The nucleic acids of the invention may also include an RNA analog or a nucleic acid complementary to sequences of the invention. In other embodiments, the nucleic acid may be a fragment of one of the above sequences, such as a fragment that is at least 5, 10, 15, 20 or 25 nucleotides in length and that hybridizes under stringent conditions to a genomic KCS2 DNA encoding the nucleic acid sequence. As used herein, the term xe2x80x9cgenomic sequencexe2x80x9d includes either of the strands of a nucleic acid molecule found in the genome of an organism or cell.
In another aspect, the nucleic acids of the invention may include a DNA coding sequence encoding an enzyme of the invention, operably linked to a promoter. Promoters of the invention may be tissue-specific or have specific developmental timing. A promoter of the invention may have a nucleotide sequence substantially identical to all or part of the KCS2 promoter region disclosed herein. Promoters of the invention may be operably linked to alternative DNAs, such as agronomically important nucleic acid sequences.
In one aspect, the invention provides methods for altering the VLCFA, fatty acid or lipid content in a plant or plant tissue, for example by introducing into a plant cell, capable of being transformed and regenerated to a whole plant, a nucleic acid of the invention. Where such nucleic acids are effective for altering the levels of VLCFAs in a plant; a plant containing the nucleic acid of the invention may be recovered having an altered phenotype.